Overexpression of
keratin 16 has been observed in keratinocytes in those
skin diseases characterized by hyperproliferation such as
psoriasis. Therefore,
keratin 16 is usually referred to as a disease-associated
keratin. In the present study, we found that
epidermal growth factor (
EGF) increased the expression of
keratin 16 mRNA and
protein synthesis in a time-dependent manner in HaCaT cells. Reporter assays revealed that the
EGF response region was in the range of -162 to -114 bp. Disruption of the Sp1 site (-127 to -122 bp) and the AP1 site (-148 to -142 bp) of the
keratin 16 promoter by site-directed mutagenesis significantly inhibited
keratin 16 promoter activity induced by
EGF. Furthermore,
keratin 16 gene expression induced by Ras activation was also regulated in the same manner as the
EGF response. By using the
DNA affinity precipitation assay in HaCaT and SL2 cells, Sp1 directly interacted with the Sp1 site of the promoter, and c-Jun and c-Fos precipitated with the Sp1
oligonucleotide was attributable to the interaction between the Sp1 and AP1
proteins. Moreover, cotransfection assays revealed that Sp1 acted synergistically with c-Jun to activate
keratin 16. The coactivators p300/CBP could collaborate with Sp1 and c-Jun in the activation of
keratin 16 promoter, and
EGF-induced promoter activation was blocked by the viral
oncoprotein E1A. Taken together, these results suggest that Sp1 and AP1 sites in the essential promoter region are critical for
EGF response, and Sp1 showed a functional cooperation with c-Jun and coactivators p300/CBP in driving the transcriptional regulation of
EGF-induced
keratin 16 gene expression.